Increase of cAMP-dependent protein kinase type II has been assessed as an early event in hormone-dependent mammary tumor regression. In this study we showed, by immunological techniques, the appearance of new species of the regulatory subunits of cAMP-dependent protein kinase type II in the nuclei of regressing as compared to growing MCF-7 tumors in nude mice. We raised monospecific antibodies against the regulatory subunits (R) of type I (RI) and type II (RII) of cAMP-dependent protein kinases derived from bovine skeletal muscle and bovine heart, respectively. The antibodies were affinity purified using glutaraldehyde cross-linked immuno-absorbents. In the radio-immunoassay, R of MCF-7 tumor cross-reacted with bovine anti-RI and RII antibodies as the bovine antigens, suggesting and immunological similarity between R of human MCF-7 tumor and bovine R. In the nuclear extracts of growing MCF-7 tumors, the anti-RI and RII antibodies, by immunoprecipitation, detected 47,000-dalton, and 44,000- and 35,000-dalton cAMP receptor proteins, respectively. Following estrogen-withdrawal, new species of cAMP receptor proteins with m.w. of 50,000 and 52,000 appeared in the nuclei of regressing tumors. The 50,000 and 52,000-dalton proteins were specifically precipitated by the anti-RII antibody but not with the RI antibody. Concomitant with the appearance of 50,000- and 52,000-dalton RII was the disappearance of the 35,000-dalton RII from the nuclei. Indirect immunofluorescence revealed that during regression of MCF-7 tumors, the intensity of immunofluorescence of RII dramatically increased in the nucleoli. The results suggest the regulatory role of type II cAMP-dependent protein kinase in mammary cancer regression.